Plasmidic gene, ways of acquiring this gene, the amylolytic enzyme it encodes and its application

ABSTRACT

The object of the invention is a novel plasmidic gene encoding an amylolytic enzyme which after introduction into a microbial strain, favorably bacterial, especially  Lactococcus lactis , enables the production of the encoded amylolytic enzyme, the ways of acquiring this gene and the industrial application of the enzyme it encodes.

BACKGROUND OF THE INVENTION

Lactic acid bacteria are capable of utilizing many different sugars as a source of carbon and energy (De Vos W. 1996. Antonie van Leeuwenhoek 70: 223). In a significant majority these sugars are monosaccharides or disaccharides. The ability of lactic acid bacteria to catabolize carbohydrates is widely used in biotechnological processes linked with food production (Libudzisz Z., Walczak P. and Bardowski J. (ed.), 1998. Lactic Acid Bacteria—classification, metabolism, genetics, application. (In polish) Monographies, Lódź; Aleksandrzak T., Kowalczyk M., Kok J., Bardowski J. 2000, Food Biotechnol. 17: 61, Elsevier Science B. V., Amsterdam). One of these processes is the production of fermented milk products. The main sugar in milk is lactose, which catabolism is a feature rather commonly present among lactic acid bacteria (Bardowski J. 1995. Przeglad Mleczarski 11: 315; Van Rooijen R. J. 1992. Lactose catabolism in Lactococcus lactis. Ph.D thesis, Wageningen, The Netherlands).

Yet another natural source, besides milk, of food production are plants. These biotechnological processes are frequently conducted with the use of lactic acid bacteria. However, in plant material, differently than in milk, polysaccharides such as cellobiose or starch are present. Thus, in the microbial conversion of plant material these microbials are incorporated which have either cellulolytic or amylolytic abilities. Lactic acid bacteria lack the ability to degrade cellulose.

However, the ability to degrade starch is, in this group of bacteria, a feature limited basically to a few strains of the Lactobacillus genus (cit. after Giraud E., A. Chapailler and M. Raimbault. 1994. Appl. Environ. Microbiol. 60: 4319). Some of these bacteria, belonging to the L. plantarum, L. amylophilus, or L. amylovorus species produce α-amylase (Fitzsimons A. and O'Connell. 1994. FEMS Microbiol. Letts. 116:137; Pompeyo C C., Gomez M S., Gasparian S., J. Morlon-Guyot 1993. Appl. Microbiol. Biotechnol. 40: 266).

It has been determined that besides strains from Lactobacillus genus, some strains of Lactococcus genus, naturally existing in the environment, also demonstrate this ability since strains which degrade starch were found among Lactococcus strains isolated from natural habitats (Domań M., E. Czerniec, Z. Targoński and J. Bardowski. (2000), Food Biotechnology 17:67, Elsevier Science B.V., Amsterdam).

Unexpectedly, it has been discovered that also other strains, especially from the Lactococcus lactis species, after introducing a specific gene structure according to the invention, are capable of producing a novel amylolytic enzyme which is encoded by this gene and which is secreted into the environment.

SUMMARY OF THE INVENTION

This invention is directed to a novel plasmidic gene encoding an amylolytic enzyme which after introduction into a microbial strain, favorably bacterial, especially Lactococcus lactis, enables the production of the encoded amylolytic enzyme, as well as ways of obtaining this gene and the industrial application of the enzyme it encodes.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The novel plasmidic gene encoding the novel amylolytic enzyme secreted into the environment, according to the invention has the following nucleotide sequence (top strand: SEQ. ID NO. 1 and bottom strand: SEQ. ID NO. 2):

5′ ATGAAAAAAACAAAATTAAGAATTTGTCTTTTCCCAATCGTACTGCTTATCAGTTTTACA    1 ---+---------+---------+---------+---------+---------+------   60 TACTTTTTTTGTTTTAATTCTTAAACAGAAAAGGGTTAGCATGACGAATAGTCAAAATGT TTGCTATCCAATGGAACTGGTAAACAGATTGTTTTTGCTACATCTTCATCAGAATATGCT   61 ---+---------+---------+---------+---------+---------+------  120 AACGATAGGTTACCTTGACCATTTGTCTAACAAAAACGATGTAGAAGTAGTCTTATACGA ACACAAGCTCTGTCTGACGAGAAAAATGCCACACAAAACAATGATTTTACATCTTTTGAT  121 ---+---------+---------+---------+---------+---------+------  180 TGTGTTCGAGACAGACTGCTCTTTTTACGGTGTGTTTTGTTACTAAAATGTAGAAAACTA ATAAATGGGCCTATGAAGGGACAGATTTGGGATTTAACTATTCAACAACCAGTACTACT  181 ---+---------+---------+---------+---------+---------+------  240 TATTTTACCCGGATACTTCCCTGTCTAAACCCTAAATTGATAAGTTGTTGGTCATGATGA TTCAAAATCTGGTCACCAACTGCGACGAGCGTGCAGCTGATAAGTTATGGGACGAATACC  241 ---+---------+---------+---------+---------+---------+------  300 AAGTTTTAGACCAGTGGTTCACGCTGCTCGCACGTCGACTATTCAATACCCTGCTTATGG AATCCGACAGCGGCACAGGTCTCAGCGAAAGCAATGACACGCGGGACATCAGCGACACCA  301 ---+---------+---------+---------+---------+---------+------  360 TTAGGCTGTCGCCGTGTCCAGAGTCGCTTTCGTTACTGTGCGCCCTGTAGTCGCTGTGGT ACTAATCACGCAACCAATACGATTGGGGTGTGGACTTTGACCGTTCCTGGCAATCAAAAT  361 ---+---------+---------+---------+---------+---------+------  420 TGATTAGTGCGTTGGTTATGCTAACCCCACACCTGAAACTGGCAAGGACCGTTAGTTTTA GGCATGGTTTATGCTTACAAACTAACTTTTGCTGACGGAACCGTTAGTGATTATGCTGGG  421 ---+---------+---------+---------+---------+---------+------  480 CCGTACCAAATACCAATGTTTGATTGAAAACGACTGCCTTGGCAATCACTAATACGACCC TCAACTTATGGAACACTTTCTACAAGTTCTGTCAGCAATACAACCAATGATCCTTATTCT  481 ---+---------+---------+---------+---------+---------+------  540 AGTTGAATACCTTGTGAAAGATGTTCAAGACAGTCGTTATGTTGGTTACTAGGAATAAGA ATTGCGACGACACAAGGTGGAAATCGTTCAGTTGTCGAATCGTCTGCAAACCTCGCGTCA  541 ---+---------+---------+---------+---------+---------+------  600 TAACGCTGCTGTGTTCCACCTTTAGCAAGTCAACAGCTTAGCAGACGTTTGGAGCGCAGT AATCTGGCACTTGCGCAAGGCAAATCTGCAACTTGGCGGGTAGCAAGTCCAACACAAGCC  601 ---+---------+---------+---------+---------+---------+------  660 TTAGACCGTGAACGCGTTCCGTTTAGACGTTGAACCCCCCATCGTTCAGCTTGTGTTCGG ATCGTTGACGAACTACATATTCGTGATTTTACAAGCTCATCAACATCTGGTGTTTCGGCT  661 ---+---------+---------+---------+---------+---------+------  720 TAGCAACTGCTTGATGTATAAGCACTAAAATGTTCGAGTAGTTGTAGACCACAAAGCCGA GGTAATCGTGGTAAGTTTCTCGGAGTGATTCAGTCAGGAACAACTGATCCAAATACAGGT  721 ---+---------+---------+---------+---------+---------+------  780 CCATTAGCACCATTCAAAGAGCCTCACTAAGTCAGTCCTTGTTGACTAGGTTTATGTCCA ACAGCGACTGGACTTGATTATCTGAAAAACGAGGGTTTTAACTACATTCAACTTCATGCC  781 ---+---------+---------+---------+---------+---------+------  840 TGTCGCTGACCTGAACTAATAGACTTTTTGCTCCCAAAATTGATGTAAGTTGAAGTACGG AGCCAGTCAGTATGCGTCGGTCAACGAAGCCGGAACCGTACTACTGCGCAACCGAATAAC  841 ---+---------+---------+---------+---------+---------+------  900 TCGGTCAGTCATACGCAGCCAGTTGCTTCGCCCTTGGCATGATGACGCGTTGGCTTATTG TTCAACTGGGGATATGACCCACAAATGAAATGGTACCAGAGCGAATATGCCAGTAATTCT  901 ---+---------+---------+---------+---------+---------+------  960 AAGTTGACCCCTATACTGGGTGTTTACTTTACCATGGTCTCGCTTATACGGTCATTAAGA GTCAATCCTGTCACACGTATTAATGAAATGAAAGAAATGGTACAAGGCTTGCATACAAAT  961 ---+---------+---------+---------+---------+---------+------ 1020 CAGTTAGGACAGTGTGCATAATTACTTTACTTTCTTTACCATGTTCCGAACGTATGTTTA GGGATTAGCGTGGTAATGGATATGGTTCTCAATCACGTCTATAGTCAGTCAGCATCCGCT 1021 ---+---------+---------+---------+---------+---------+------ 1080 CCCTAATCGCACCATTACCTATACCAAGAGTTAGTGCAGATATCAGTCAGTCGTAGGCGA TTTGAAAAAGCCGAGCCGGGCTATTATTTCCGCAAAAATACGCAATCTGGGTGTGGCAAT 1081 ---+---------+---------+---------+---------+---------+------ 1140 AAACTTTTTCGGCTCGGCCCGATAATAAAGGCGTTTTTATGCGTTAGACCCACACCGTTA GATACGGCGAGCAACCACGAAATGTTTGGTAAATACATTATTGACTCTGTCACTTACTGG 1141 ---+---------+---------+---------+---------+---------+------ 1200 CTATGCCGCTCGTTGGTGCTTTACAAACCATTTATGTAATAACTGAGACAGTGAATGACC GCGAAAAATTATGATATTGACGGCTTCCGTTTTGATGAAATGACACTTTTAGACAGCACG 1201 ---+---------+---------+---------+---------+---------+------ 1260 CGCTTTTTAATACTATAACTGCCGAAGGCAAAACTACTTTACTGTGAAAATCTGTCGTGC ACTATGAATAAACTGCGTGCCGCACTGACAGCACTTGACCCACATATTATCATGTATGGC 1261 ---+---------+---------+---------+---------+---------+------ 1320 TGATACTTATTTGACGCACGGCGTGACTGTCGTGAACTGGGTGTATAATAGTACATACCG GAAGGTTGGGGCGATTCTAATGCAAATAATATTCCTGAAACGTCTATCAATAATTACAAA 1321 ---+---------+---------+---------+---------+---------+------ 1380 CTTCCAACCCCGCTAAGATTACGTTTATTATAAGGACTTTGCAGATAGTTATTAATGTTT AATGTTCCTGGTATTGGTTTTTTCAATCCTGGTGAACGTGACGCCATCAGTAATAATGGA 1381 ---+---------+---------+---------+---------+---------+------ 1440 TTACAAGGACCATAACCAAAAAAGTTAGGACCACTTGCACTGCGGTAGTCATTATTACCT GGTTCTGCAGGTGGATTCGCAGCAGGAAATACGGCAAGTACTATAACGGTCGCAGGAGCA 1441 ---+---------+---------+---------+---------+---------+------ 1500 CCAAGACGTCCACCTAAGCGTCGTCCTTTATGCCGTTCATGATATTGCCAGCGTCCTCGT CTTTTGGCTTCTGCAGGTTGGAACGGAAATGGCACAGTGCAAGCTTTTTTGACGCCAAGC 1501 ---+---------+---------+---------+---------+---------+------ 1560 GAAAACCGAAGACCTCCAACCTTGCCTTTACCGTGTCACGTTCGAAAAAACTGCGGTTCG CAGTCTATCAATTATGTTGAATGTCATGATAGCTTCACCTTAAATGACTCACTCTGGTCA 1561 ---+---------+---------+---------+---------+---------+------ 1620 GTCAGATAGTTAATACAACTTACAGTACTATCGAAGTGGAATTTACTGAGTGAGACCAGT GCTGATCCGAATGATTCTGTGGCGACACATCAAGCGCGAGTGACGCTCGCAAATGCCACG 1621 ---+---------+---------+---------+---------+---------+------ 1680 CGACTAGGCTTACTAAGACACCGCTGTGTAGTTCGCGCTCACTGCGAGCGTTTACGGTGC AATATTCTTGCAAACGGTGTGACATTTATGGAAACAGGACAGGAGTTTGACCAGTCCAAA 1681 ---+---------+---------+---------+---------+---------+------ 1740 TTATAAGAACGTTTGCCACACTGTAAATACCTTTGTCCTGTCCTCAAACTGGTCAGGTTT TTAGTCAATCCGTCAAATCTGACGCCGCTCTCCCCTACACAGACACAGGCTTATCAATCG 1741 ---+---------+---------+---------+---------+---------+------ 1800 AATCAGTTAGGCAGTTTAGACTGCGGCGAGAGGGGATGTGTCTGTGTCCGAATAGTTAGC GGTAGTATGGAAAAACCTGCATGGTATCCTGCGTCATGGGATACTGCCAAAAATTCATAC 1801 ---+---------+---------+---------+---------+---------+------ 1860 CCATCATACCTTTTTGGACGTACCATAGGACGCAGTACCCTATGACGGTTTTTAAGTATG AATGGACTTTTTGGCTTGGCTAGTAATGGTACTTACTATGGCAATTATTGGCCAGGAAGT 1861 ---+---------+---------+---------+---------+---------+------ 1920 TTACCTGAAAAACCGAACCGATCATTACCATGAATGATACCGTTAATAACCGGTCCTTCA AATCTCTACACCCCAGTAGTTGCGGGGGATGTAGTCAATGCCATGAATTGGGATAATGTC 1921 ---+---------+---------+---------+---------+---------+------ 1980 TTAGAGATGTGGGGTCATCAACGCCCCCTACATCAGTTACGGTACTTAACCCTATTACAG AAGGACAATCAAAATGCTGTTAATTTTATTGGAAATTTGATGAAATTTAAAAAATCCAAT 1981 ---+---------+---------+---------+---------+---------+------ 2040 TTCCTGTTAGTTTTACGACAATTAAAATAACCTTTAAACTACTTTAAATTTTTTAGGTTA CCGCAATTTTGGCCTGATGATTATAGCAAACTTGCTTGGACTCCTACCAGTATAGGTGTA 2041 ---+---------+---------+---------+---------+---------+------ 2100 GGCGTTAAAACCGGACTACTAATATCGTTTGAACGAACCTGAGGATGGTCATATCCACAT GAAAATGTCACAAATGCGTCAAACGGAGTGATCACAGAAGAATTAACATCGGGTGCGACC 2101 ---+---------+---------+---------+---------+---------+------ 2160 CTTTTACAGTGTTTACGCAGTTTGCCTCACTAGTGTCTTCTTAATTGTAGCCCACGCTGG AAATATTTAGTCATATTGAATGCTAGCGGTAATTCGGTCAAAATTGGTCAAGGTGGTCAG 2161 ---+---------+---------+---------+---------+---------+------ 2220 TTTATAAATCAGTATAACTTACGATCGCCATTAAGCCAGTTTTAACCAGTTCCACCAGTC TTTTATGGGGTGTCAAATTTGACAGGTAAAACTGTGATAATTTCTGACGATAGTAGCTTA 2221 ---+---------+---------+---------+---------+---------+------ 2280 AAAATACCCCACAGTTTAAACTGTCCATTTTGACACTATTAAAGACTGCTATCATCGAAT ACAGCTAATCAAGTTTTGAATAGCTCTGTCACTCTGTCAAATTTGACAGTCACCGTTATC 2281 ---+---------+---------+---------+---------+---------+------ 2340 TGTCGATTAGTTCAAAACTTATCGAGACAGTGAGACAGTTTAAACTGTCAGTGGCAATAG CAACTTTCTAAATAA 2341 ---+---------+- 2355             3′ GTTGAAAGATTTATT

The novel gene according to the invention favorably has at its 5′ end an additional nucleotide sequence which together give the following nucleotide structure: (top strand: SEQ. ID NO. 3 and bottom strand: SEQ. ID NO. 4)

TTACGTTAGCTGGAGAATCCACCGTGTGGAGCCTCCGTCTCACGGTTGCTCCGTGTTACG    1 ---------+---------+---------+---------+---------+---------+   60 AATGCAATCGACCTCTTAGGTGGCACACCTCGGAGGCAGAGTGCCAACGAGGCACAATGC GTAATTCCGCTGCTATAAAGTAATAACTAATAACAAATTAAATAGCAGTATAAAATTTTG   61 ---------+---------+---------+---------+---------+---------+  120 CATTAAGGCGACGATATTTCATTATTGATTATTGTTTAATTTATCGTCATATTTTAAAAC ATAATTTGAAGATTATTTTAATATTTACCTGAATAACACTTTAGAAAACTGAAATATCTA  121 ---------+---------+---------+---------+---------+---------+  180 TATTAAACTTCTAATAAAATTATAAATGGACTTATTGTGAAATCTTTTGACTTTATAGAT TAACTCCAGATCAATAGTGGAAATACGTACAATTTGATATAAGAGATTTTTGTATCAAAA  181 ---------+---------+---------+---------+---------+---------+  240 ATTGAGGTCTAGTTATCACCTTTATGCATGTTAAACTATATTCTCTAAAAACATAGTTTT AAAATTTTTTTAAACAAAATGCACTAAAAGATTTTTTTGTGTTGCTGAGAGGTTGCAAAA  241 ---------+---------+---------+---------+---------+---------+  300 TTTTAAAAAAATTTGTTTTACGTGATTTTCTAAAAAAACACAACGACTCTCCAACGTTTT TGATACCGCAAGGTATATAATGGTTACGTAATTAGTGCAAGCGCTTGCATTTTTTGTGAA  301 ---------+---------+---------+---------+---------+---------+  360 ACTATGGCGTTCCATATATTACCAATGCATTAATCACGTTCGCGAACGTAAAAACACTT AAAAGTCCTTTTATTTATTTATCTTATTGCTTTGAAAGATATTGTTAAAAATAAATAAAA  361 ---------+---------+---------+---------+---------+---------+  420 TTTTCAGGAAAATAAATAAATAGAATAACGAAACTTTCTATAACAATTTTTATTTATTTT TCGTTATAAACTCAATATAATAAGGAGAGCTAAATAATGAAAAAAACAAAATTAAGAATT  421 ---------+---------+---------+---------+---------+---------+  480 AGCAATATTTGAGTTATATTATTCCTCTCGATTTATTACTTTTTTTGTTTTAATTCTTAA TGTCTTTTCCCAATCGTACTGCTTATCAGTTTTACATTGCTATCCAATGGAACTGGTAAA  481 ---------+---------+---------+---------+---------+---------+  540 ACAGAAAAGGGTTAGCATGACGAATAGTCAAAATGTAACGATAGGTTACCTTGACCATTT CAGATTGTTTTTGCTACATCTTCATCAGAATATGCTACACAAGCTCTGTCTGACGAGAAA  541 ---------+---------+---------+---------+---------+---------+  600 GTCTAACAAAAACGATGTAGAAGTAGTCTTATACGATGTGTTCGAGACAGACTGCTCTTT AATGCCACACAAAACAATGATTTTACATCTTTTGATATAAAATGGGCCTATGAAGGGACA  601 ---------+---------+---------+---------+---------+---------+  660 TTACGGTGTGTTTTGTTACTAAAATGTAGAAAACTATATTTTACCCGGATACTTCCCTGT GATTTGGGATTTAACTATTCAACAACCAGTACTACTTTCAAAATCTGGTCACCAACTGCG  661 ---------+---------+---------+---------+---------+---------+  720 CTAAACCCTAAATTGATAAGTTGTTGGTCATGATGAAAGTTTTAGACCAGTGGTTGACGC ACGAGCGTGCAGCTGATAAGTTATGGGACGAATACCAATCCGACAGCGGCACAGGTCTCA  721 ---------+---------+---------+---------+---------+---------+  780 TGCTCGCACGTCGACTATTCAATACCCTGCTTATGGTTAGGCTGTCGCCGTGTCCAGAGT GCGAAAGCAATGACACGCGGGACATCAGCGACACCAACTAATCACGCAACCAATACGATT  781 ---------+---------+---------+---------+---------+---------+  840 CGCTTTCGTTACTGTGCGCCCTGTAGTCGCTGTGGTTGATTAGTGCGTTGGTTATGCTAA GGGGTGTGGACTTTGACCGTTCCTGGCAATCAAAATGGCATGGTTTATGCTTACAAACTA  841 ---------+---------+---------+---------+---------+---------+  900 CCCCACACCTGAAACTGGCAAGGACCGTTAGTTTTACCGTACCAAATACGAATGTTTGAT ACTTTTGCTGACGGAACCGTTAGTGATTATGCTGGGTCAACTTATGGAACACTTTCTACA  901 ---------+---------+---------+---------+---------+---------+  960 TGAAAACGACTGCCTTGGCAATCACTAATACGACCCAGTTGAATACCTTGTGAAAGATGT AGTTCTGTCAGCAATACAACCAATGATCCTTATTCTATTGCGACGACACAAGGTGGAAAT  961 ---------+---------+---------+---------+---------+---------+ 1020 TCAAGACAGTCGTTATGTTGGTTACTAGGAATAAGATAACGCTGCTGTGTTCCACCTTTA CGTTCAGTTGTCGAATCGTCTGCAAACCTCGCGTCAAATCTGGCACTTGCGCAAGGCAAA 1021 ---------+---------+---------+---------+---------+---------+ 1080 GCAAGTCAACAGCTTAGCAGACGTTTGGAGCGCAGTTTAGACCGTGAACGCGTTCCGTTT TCTGCAACTTGGCGGGTAGCAAGTCCAACACAAGCCATCGTTGACGAACTACATATTCGT 1081 ---------+---------+---------+---------+---------+---------+ 1140 AGACGTTGAACCGCCCATCGTTCAGGTTGTGTTCGGTAGCAACTGCTTGATGTATAAGCA GATTTTACAAGCTCATCAACATCTGGTGTTTCGGCTGGTAATCGTGGTAAGTTTCTCGGA 1141 ---------+---------+---------+---------+---------+---------+ 1200 CTAAAATGTTCGAGTAGTTGTAGACCACAAAGCCGACCATTAGCACCATTCAAAGAGCCT GTGATTCAGTCAGGAACAACTGATCCAAATACAGGTACAGCGACTGGACTTGATTATCTG 1201 ---------+---------+---------+---------+---------+---------+ 1260 CACTAAGTCAGTCCTTGTTGACTAGGTTTATGTCCATGTCGCTGACCTGAACTAATAGAC AAAAACGAGGGTTTTAACTACATTCAACTTCATGCCAGCCAGTCAGTATGCGTCGGTCAA 1261 ---------+---------+---------+---------+---------+---------+ 1320 TTTTTGCTCCCAAAATTGATGTAAGTTGAAGTACGGTCGGTCAGTCATACGCAGCCAGTT CGAAGCGGGAACCGTACTACTGCGCAACCGAATAACTTCAACTGGGGATATGACCCACAA 1321 ---------+---------+---------+---------+---------+---------+ 1380 GCTTCGCCCTTGGCATGATGACGCGTTGGCTTATTGAAGTTGACCCCTATACTGGGTGTT ATGAATGGTACCAGAGCGAATATGCCAGTAATTCTGTCAATCCTGTCACACGTATTAAT 1381 ---------+---------+---------+---------+---------+---------+ 1440 TACTTTACCATGGTCTCGCTTATACGGTCATTAAGACAGTTAGGACAGTGTGCATAATTA GAAATGAAAGAAATGGTACAAGGCTTGCATACAAATGGGATTAGCGTGGTAATGGATATG 1441 ---------+---------+---------+---------+---------+---------+ 1500 CTTTACTTTCTTTACCATGTTCCGAACGTATGTTTACCCTAATCGCACCATTACCTATAC GTTCTCAATCACGTCTATAGTCAGTCAGCATCCGCTTTTGAAAAAGCCGAGCCGGGCTAT 1501 ---------+---------+---------+---------+---------+---------+ 1560 CAAGAGTTAGTGCAGATATCAGTCAGTCGTAGGCGAAAACTTTTTCGGCTCGGCCCGATA TATTTCCGCAAAAATACGCAATCTGGGTGTGGCAATGATACGGCGAGCAACCACGAAATG 1561 ---------+---------+---------+---------+---------+---------+ 1620 ATAAAGGCGTTTTTATGCGTTAGACCCACACCGTTACTATGCCGCTCGTTGGTGCTTTAC TTTGGTAAATACATTATTGACTCTGTCACTTACTGGGCGAAAAATTATGATATTGACGGC 1621 ---------+---------+---------+---------+---------+---------+ 1680 AAACCATTTATGTAATAACTGAGACAGTGAATGACCCGCTTTTTAATACTATAACTGCCG TTCCGTTTTGATGAAATGACACTTTTAGACAGCACGACTATGAATAAACTGCGTGCCGCA 1681 ---------+---------+---------+---------+---------+---------+ 1740 AAGGCAAAACTACTTTACTGTGAAAATCTGTCGTGCTGATACTTATTTGACGCACGGCGT CTGACAGCACTTGACCCACATATTATCATGTATGGCGAAGGTTGGGGCGATTCTAATGCA 1741 ---------+---------+---------+---------+---------+---------+ 1800 GACTGTCGTGAACTGGGTGTATAATAGTACATACCGCTTCCAACCCCGCTAAGATTACGT AATAATATTCCTGAAACGTCTATCAATAATTACAAAAATGTTCCTGGTATTGGTTTTTTC 1801 ---------+---------+---------+---------+---------+---------+ 1860 TTATTATAAGGACTTTGCAGATAGTTATTAATGTTTTTACAAGGACCATAACCAAAAAAG AATCCTGGTGAACGTGACGCCATCAGTAATAATGGAGGTTCTGCAGGTGGATTCGCAGCA 1861 ---------+---------+---------+---------+---------+---------+ 1920 TTAGGACCACTTGCACTGCGGTAGTCATTATTACCTCCAAGACGTCCACGTAAGCGTCGT GGAAATACGGCAAGTACTATAACGGTCGCAGGAGCACTTTTGGCTTCTGGAGGTTGGAAC 1921 ---------+---------+---------+---------+---------+---------+ 1980 CCTTTATGCCGTTCATGATATTGCCAGCGTCCTCGTGAAAACCGAAGACCTCCAACCTTG GGAAATGGCACAGTGCAAGCTTTTTTGACGCCAAGCCAGTCTATCAATTATGTTGAATGT 1981 ---------+---------+---------+---------+---------+---------+ 2040 CCTTTACCGTGTCACGTTCGAAAAAACTGCGGTTCGGTCAGATAGTTAATACAACTTACA GATGATAGCTTCACCTTAAATGACTCACTCTGGTCAGCTGATCCGAATGATTCTGTGGCG 2041 ---------+---------+---------+---------+---------+---------+ 2100 GTACTATCGAAGTGGAATTTACTGAGTGAGACCAGTCGACTAGGCTTACTAAGACACCGC ACACATCAAGCGCGAGTGACGCTCGCAAATGCCACGAATATTCTTGCAAACGGTGTGACA 2101 ---------+---------+---------+---------+---------+---------+ 2160 TGTGTAGTTCGCGCTCACTGCGAGCGTTTACGGTGCTTATAAGAACGTTTGCCACACTGT TTTATGGAAACAGGACAGGAGTTTGACCAGTCCAAATTAGTCAATCCGTCAAATCTGACG 2161 ---------+---------+---------+---------+---------+---------+ 2220 AAATACCTTTGTCCTGTCCTCAAACTGGTCAGGTTTAATCAGTTAGGCAGTTTAGACTGC CCGCTCTCCCCTACACAGACACAGGCTTATCAATCGGGTAGTATGGAAAAACCTGCATGG 2221 ---------+---------+---------+---------+---------+---------+ 2280 GGCGAGAGGGGATGTGTCTGTGTCCGAATAGTTAGCCCATCATACCTTTTTGGACGTACC TATCCTGCGTCATGGGATACTGCCAAAAATTCATACAATGGACTTTTTGGCTTGGCTAGT 2281 ---------+---------+---------+---------+---------+---------+ 2340 ATAGGACGCAGTACCCTATGACGGTTTTTAAGTATGTTACCTGAAAAACCGAACCGATCA AATGGTACTTACTATGGCAATTATTGGCCAGGAAGTAATCTCTACACCCCAGTAGTTGCG 2341 ---------+---------+---------+---------+---------+---------+ 2400 TTACCATGAATGATACCGTTAATAACCGGTCCTTCATTAGAGATGTGGGGTCATCAACGC GGGGATGTAGTCAATGCCATGAATTGGGATAATGTCAAGGACAATCAAAATGCTGTTAAT 2401 ---------+---------+---------+---------+---------+---------+ 2460 CCCCTACATCAGTTACGGTACTTAACCCTATTACAGTTCCTGTTAGTTTTACGACAATTA TTTATTGGAAATTTGATGAAATTTAAAAAATCCAATCCGCAATTTTGGCCTGATGATTAT 2461 ---------+---------+---------+---------+---------+---------+ 2520 AAATAACCTTTAAACTACTTTAAATTTTTTAGGTTAGGCGTTAAAACCGGACTACTAATA AGCAAACTTGCTTGGACTCCTACCAGTATAGGTGTAGAAAATGTCACAAATGCGTCAAAC 2521 ---------+---------+---------+---------+---------+---------+ 2580 TCGTTTGAACGAACCTGAGCATGGTCATATCCACATCTTTTACAGTGTTTACGCAGTTTG GGAGTGATCACAGAAGAATTAACATCGGGTGCGACCAAATATTTAGTCATATTGAATGCT 2581 ---------+---------+---------+---------+---------+---------+ 2640 CCTCACTAGTGTCTTCTTAATTGTAGCCCACGCTGGTTTATAAATCAGTATAACTTACGA AGCGGTAATTCGGTCAAAATTGGTCAAGGTGGTCAGTTTTATGGGCTGTCAAATTTGACA 2641 ---------+---------+---------+---------+---------+---------+ 2700 TCGCCATTAAGCCAGTTTTAACCAGTTCCACCAGTCAAAATACCCCACAGTTTAAACTGT GGTAAAACTGTGATAATTTCTGACGATAGTAGCTTAACAGCTAATCAAGTTTTGAATAGC 2701 ---------+---------+---------+---------+---------+---------+ 2760 CCATTTTGACACTATTAAAGACTGCTATCATCGAATTGTCGATTAGTTCAAAACTTATCG TCTGTCACTCTGTCAAATTTGACAGTCACCGTTATCCAACTTTCTAAATAA 2761 ---------+---------+---------+---------+---------+- 2811 AGACAGTGAGACAGTTTAAACTGTCAGTGGCAATAGGTTGAAAGATTTATT

Ways of producing the novel plasmidic gene encoding the novel amylolytic enzyme which is secreted into the environment, according to the invention, are as follows: the plasmid DNA, previously isolated from the bacterial strain, favorably belonging to Lactococcus genus, encoding the amylolytic enzyme, is digested with restriction enzymes, favorably EcoRI, SacII, SalI, SmaI, SpeI, XhoI or XbaI. Subsequently, the excised fragment, favorably of size not smaller than 3.0 kb is ligated with a plasmid capable of replicating in bacterial cells, favorably those of Lactococcus genus, especially pIL253, pGKV210 or pIL252 also digested previously with restriction enzymes, favorably with EcoRI, SalI, or SmaI, after which the two DNA fragments are recombined together and introduced by means of electroporation into bacterial cells, which are grown in a known manner, and from the cultivated population, cells possessing the novel gene encoding the novel amylolytic enzyme are isolated in a known way.

In the method according to the invention bacterial strains into which the recombined DNA is introduced are favorably used Lactococcus lactis MG1363, Lactococcus lactis IL1403 or Lactococcus lactis IBB140.

In the method according to the invention colonies producing the amylolytic enzyme and carrying the novel gene are positively separated by inoculation into fresh liquid medium with antibiotic and starch if necessary.

The novel enzyme secreted into the environment, encoded by the novel plasmidic gene according to the invention, has the following amino acid structure (SEQ. ID NO. 5):

  1 MKKTKLRICL FPIVLLISFT LLSNGTGKQI VFATSSSEYA TQALSDEKNA  51 TQNNDFTSFD IKWAYEGTDL GFNYSTTSTT FKIWSPTATS VQLISYGTNT 101 NPTAAQVSAK AMTRGTSATP TNHATNTIGV WTLTVPGNQN GMVYAYKLTF 151 ADGTVSDYAG STYGTLSTSS VSNTTNDPYS IATTQGGNRS VVESSANLAS 201 NLALAQGKSA TWRVASPTQA IVDELHIRDF TSSSTSGVSA GNRGKFLGVI 251 QSGTTDPNTG TATGLDYLKN EGFNYIQLHA SQSVCVGQRS GNRTTAQPNN 301 FNWGYDPQMK WYQSEYASNS VNPVTRINEM KEMVQGLHTN GISVVMDMVL 351 NHVYSQSASA FEKAEPGYYF RKNTQSGCGN DTASNHEMFG KYIIDSVTYW 401 AKNYDIDGFR FDEMTLLDST TMNKLRAALT ALDPHIIMYG EGWGDSNANN 451 IPETSINNYK NVPGIGFFNP GERDAISNNG GSAGGFAAGN TASTITVAGA 501 LLASGGWNGN GTVQAFLTPS QSINYVECHD SFTLNDSLWS ADPNDSVATH 551 QARVTLANAT NILANGVTFM ETGQEFDQSK LVNPSNLTPL SPTQTQAYQS 601 GSMEKPAWYP ASWDTAKNSY NGLFGLASNG TYYGNYWPGS NLYTPVVAGD 651 VVNAMNWDNV KDNQNAVNFI GNLMKFKKSN PQFWPDDYSK LAWTPTSIGV 701 ENVTNASNGV ITEELTSGAT KYLVILNASG NSVKIGQGGQ FYGVSNLTGK 751 TVIISDDSSL TANQVLNSSV TLSNLTVTVI QLSK

This enzyme exhibits a unique set of profitable features, especially showing activity in a wide range of acid pH from 3.5 to 5.5 as shown on graph (FIG. 1) while its optimum is at a pH 4.4. At the same time the optimal temperature of the enzyme's activity is in a range from 35°-45° C. as shown on graph (FIG. 2), and is thermally inactivated at temperatures higher than 50° C. as shown on graph (FIG. 3).

Application of the amylolytic enzyme encoded by the novel gene according to the invention, in fermentation of plant material, favorably starch, pullulan, amylose, amylopectin, feed production, obtaining glucose, lactic acid and production of probiotic specimens, probiotic-containing food, feed and fodder, according to the invention is based on the fact that its gene is introduced into the structure of the bacterial strain, favorably the strains of Lactococcus lactis IBB500, IBB501, IBB502, IBB140 and subsequently of applying the strains obtained in fermentations of plant material or milk or in biomass production.

The gene according to the invention has been sequenced and the sequence obtained indicates that it belongs to the family of pullulanase encoding genes. The analysis of the gene's promoter region as well as functional studies suggest that the expression of this gene undergoes catabolic repression.

General Characteristics of the Amylolytic Enzyme

From the comparison of features of pullulanases from studied L. lactis IBB500 and IBB502 strains it can be concluded that we are dealing with an enzyme produced in two organisms. Pullulanase deriving from both of the strains had the optimum pH of 4.4 and reached the optimal temperature at 45° C. (FIGS. 1, 2).

One should note the rather narrow pH range of the enzyme's activity between pH 3.5-5.5 as well as the fact that it is significantly lower than for pullulanases deriving from other bacteria (Ara K., Igarashi K., Saeki K., Kawai S., and S., Ito. 1992. Biosci. Biotech. Biochem. 56:62; Kim Ch., Nashiru O., J., Ko. 1996. FEMS Microbiology Letters 138:147; Takasaki Y. 1987. Agric. Biol. Chem., 51:9).

The enzyme obtained with the help of the novel gene according to the invention, shows homology to amylolytic enzymes of the pullulanase group and the highest homology was found to pullulanase from Termotoga maritima. In the amino acid sequence of pullulanase from L. lactis IBB500 four conserved motives, characteristic for many amylolytic enzymes were identified. The nucleotide sequence of pul gene was preceded by a RBS typical for Lactococcus in front of which a long 500-nucleotide non-coding region was identified.

The thorough analysis of the non-coding region upstream of the pul gene showed the presence of several putative promoter sequences. Due to the earlier observations of the effect of glucose on pullulanase production, from which was concluded that the expression of the pul gene may be regulated by catabolic repression, the promoter region was analyzed for the presence of the cre motive, characteristic for this mechanism of regulation (Weickert M. J. and S. Adhya. 1992. J. Biol. Chem. 267: 15869). It was determined that the sequence homologous to the 14-nucleotide cre sequence is present in the analyzed promoter region. This observation strongly supported the earlier hypothesis. Another argument for the validity of this hypothesis was acquired during experiments on the influence of glucose as well as starch and its derivates on the pullulanase production in the IBB500 strain. In the process it was determined that starch and its derivatives induce pullulanase production although at different levels, while in the presence of glucose the repression of production of this enzyme is observed (Domań M., E. Czerniec, Z. Targoński and J. Bardowski. 2000. Food Biotechnology 17:67, Elsevier Science B.V., Amsterdam).

HERE BELOW THE EXAMPLES OF REALIZATION ARE PRESENTED

Materials and Methods

Bacterial Strains, Growth Conditions and Plasmids Used.

Bacterial strains and plasmids used in these studies are shown in Table 1. Lactococcus lactis strains were grown in BHI medium (Oxoid, England) or M17 (Difco, USA), at the temperature of 28° C., Escherichia coli in Luria-Bertani medium (LB), at the temperature of 37° C. Where necessary for selection, the following antibiotics were used: erythromycin—5 μg/ml for L. lactis and ampicyline—100 μg/ml for E. coli.

Example I

Plasmid DNA was isolated from Lactococcus lactis IBB500 bacterial strain encoding the amylolytic enzyme. Subsequently, it was digested with EcoRI restriction enzyme, then the excised fragment not smaller that 8.0 kb was ligated with a plasmid capable of replicating in Lactococcus bacterial cells—pIL253, digested previously also with EcoRI restriction enzyme. Both DNAs were recombined with each other and introduced by the method of electroporation into Lactococcus lactis cells which were grown and from the cultivated population cells carrying the novel gene of the structure according to the invention were isolated by inoculation into fresh liquid medium with antibiotic and starch.

L. lactis cells were transformed by method of electroporation (Holo H. and I. F. Nes. 1989. Appl. Environ. Microbiol. 55: 3119).

The remaining molecular biology techniques used in the example were carried out according to the standard methodology (Sambrook J., E. F. Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2^(nd) edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

Example II

Plasmid DNA was isolated from Lactococcus lactis IBB500 bacterial strain encoding the amylolytic enzyme. Subsequently, it was digested with XhoI restriction enzyme, the excised fragment of size not smaller than 5.0 kb was ligated with a plasmid capable of replicating in Lactococcus bacterial cells—pIL253, digested previously also with XhoI. Both DNAs were then recombined with each other and introduced by method of electroporation into Lactococcus lactis cells which were grown and from the cultivated population cells carrying the novel gene of the structure according to the invention was selected by inoculation into fresh liquid medium with antibiotic and starch.

L. lactis cells were transformed by method of electroporation (Holo H. and I. F. Nes. 1989. Appl. Environ. Microbiol. 55: 3119).

The remaining molecular biology techniques used in the example were carried out according to the standard methodology (Sambrook J., E. F. Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2^(nd) edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

Example III

The structure of the nucleotide sequence of the novel gene and the amino acid sequence of the amylolytic enzyme encoded by this gene according to the invention was determined by sequencing a Pstl 1.5 kb fragment of the pIBB502 plasmid, carrying a part of the novel gene, which was previously cloned in pBluescript plasmid (Stratagene), what generated the pIBB504 plasmid. This plasmid was used to sequence the cloned fragment from both sides. Transformation of E. coli cells was carried out according to the standard method with the use of CaCl₂ (Sambrook J., E. F. Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2^(nd) edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

The complete nucleotide sequence of the fragment and of the lacking part of the novel gene encoding the amylolytic enzyme were obtained by using the sequencing method named “primer walking”. For DNA sequencing BigDye Terminator set (Promega, USA), PCR machine model 2400 (Perkin-Elmer) and sequencer ABI377 (Applied Biosystem, USA) were used. The nucleotide and amino acid sequences obtained were analyzed using the GCG programs (Genetics Computer Group. 1991. Program manual for the GCG package, version 7, April 1991. Genetic Computer Group, Madison, Wis., USA) and BLAST (Altschul S. F., W. Gish, W. Miller, E. W. Myers and D. J. Lipman. (1990), J. Mol. Biol. 215: 403).

Example IV

For determining the amylolytic activity two tests—quantitative and qualitative were applied.

Qualitative Test on Plates

Bacterial strains were plated on BHI with 0.5% starch and incubated in 28° C. for two days. Subsequently, the grown colonies were overlayed with Lugol reagent, which colors the non-degraded starch dark blue. A clear zone of non-colored medium surrounded colonies that secrete the amylolytic enzyme into the environment.

Quantitative Determination of Enzyme Activity

In this test a modified method was used to determine the activity of extracellular amylase (Nicholson W. and P. Setlow. 1990. Sporulation, germination and outgrowth, p 433. W C. R. Harwood and S. M. Cutting (ed.), Molecular Biology for Bacillus. John Wiley and Sons Ltd., Chichester, United Kingdom).

In this method the enzymatic activity was determined in the supernatant of the culture: to 200 μl of this supernatant 800 μl of the substrate (0.025% starch in 10 mM Tris-HCl-3 mM potassium acetate-25 mM CaCl₂, pH 4.4) was added and incubated for 30 min. at 37° C. The reaction was stopped by adding 400 μl of Lugol reagent and the absorption was measured at wavelength of 620 nm [A₆₂₀]. As a control non-inoculated medium was used.

One unit of amylolytic activity (1 U) was established as the amount of enzyme, which in the conditions of the test, generated the decrease of the substrate's absorption A₆₂₀ by 0.1 compared to the control.

TABLE 1 Strains and plasmids. Strains and plasmids Genotype Source Lactococcus lactis IBB 500 amy⁺ wild type strain IBB PAN collection isolated from plant material IBB 501 amy⁺ MG 1363 with this work pIBB501 IBB 502 amy⁺ derivative of MG this work 1363 with pIBB502 IBB 140 amy⁺ IBB140 with this work pIBB502 IL 1403 amy⁻, plamid-free A. Chopin, France MG 1363 amy⁻, plamid-free M. Gasson, England Eschercia coli TG1 supE thi Δ(lac-proAB) hsd (Gibson, 1984) (F′⁺ traD proAB lacl^(q) Z M15) IBB 504 amy⁻ derivative of TG1 this work with pIBB504 Plasmids pIBB500 30-kb, wild type plasmid this work with amy⁺ gene pIBB501 20-kb fragment of pIBB500 this work with amy⁺ gene cloned in pIL253 pIBB502 13-kb derivative of this work pIBB501 with amy⁺ gene pIBB504 1.5-kb Pstl fragment from this work pIBB502 in pSKII+ pBluescript SKII+ Amp^(R), vector for cloning Stratagene, USA and DNA sequencing pIL253 Ery^(R), vector for gene A. Chopin, France cloning 

1. An isolated polypeptide comprising the amino acid sequence of SEQ ID No.
 5. 2. The polypeptide according to claim 1 which is encoded by the nucleotide sequence of SEQ ID No.
 1. 